Cell communication via gap junctions involves direct cell-to-cell diffusion of ions and small cytosolic molecules, and is mediated by channels that create hydrophilic pathways across two apposed plasma membranes and a narrow extracellular space (gap). Each channel is a multimere of proteins subunits (connexins, Cx) that insulate the hydrophilic pore within the membranes and across the gap. Gap junction communication is a fundamental cellular function whose abnormality has been shown to have serious consequences for organ function such as cardiac arrhythmias, uterine malfunction at birth, Charcot-Marie-Tooth demyelinating disease (CMTX1), cardiac malformation, epileptic seizures, etc. In recent years, much has been learned on gap junction channel biochemistry, biophysics and cellular biology. Yet, crucial elements of channel structure and function are still hypothetical. This proposal is aimed at improving our understanding of the molecular basis of channel gating, channel insulation, connexin interaction, and channel lining structure, by applying a multiple approach that includes: connexin mutagenesis, expression of connexins in Xenopus oocyte pairs and small cells (RIN cells), recording of junctional conductance and single-channel activity, measurement of intracellular Ca and pH, and electron microscopy. Much of the proposal stems from our recent finding that the inner loop domain of the liver connexin (Cx32, a Cx implicated in CMTX1) plays a key role in chemical and voltage gating of gap junction channels. To understand its function in channel gating, we will first test for pH and voltage sensitivity most connexins, expressed in oocytes and RIN ells. Then, we will construct chimeras that swap cytoplasmic domains between connexins of low and high pH sensitivity, as well as connexins mutated or deleted at crucial cytoplasmic domains. Other projects will study permeability and gating of hemichannels made of wild-type and mutated connexins, the long range goal being to learn how to exploit hemichannels as vehicles for selective drug delivery to cells. By connexin mutagenesis and cysteine-substitution approach we will search for the channel lining structure and will test the gating hypothesis based on rotation of the third transmembrance domain. Our hypothesis of channel regulation by calcineurin-mediated dephosphorylation will be tested with inhibitors of kinases and phosphatases. Finally, the molecular basis of channel insulation and connexin interaction will be probed through appropriate deletions and mutations of external Cx domains.